Functionally activate neutrophils are critical to the body's innate capacity to eradicate bacterial and fungal infections. During neutrophil differentiation in the bone marrow, the nucleus becomes uniquely lobulated, which is thought to facilitate the escape of neutrophils from capillary vessels into infected tissues. In certain pathologic and genetic disorders, lobulation of the neutrophil nucleus is disrupted, and the identification of hypolobulated neutrophils in circulation is an important diagnostic indicator of myelodysplasias that often precede acute myelogenous leukemias. Recent studies have demonstrated that expression of a nuclear envelope protein called the lamin B receptor (LBR) is essential to the formation of the lobulated neutrophil nucleus. However, how LBR functions to orchestrate lobulation of the neutrophil nucleus and what role it plays in neutrophil functional activation is unknown. The long-term objective of this proposal is to understand the molecular mechanisms governing the dramatic changes in nuclear architecture that occur during neutrophil differentiation and to identify the role these changes play during neutrophil functional activation. To help attain this objective, this laboratory has generated a novel progenitor cell line that carries a genetic deficiency of LBR expression and that demonstrates defective neutrophil differentiation and functional activation. The specific aims of this project are: a) to characterize the expression of nuclear envelope-associated proteins during the differentiation of LBR-deficient neutrophils, b) to determine how disruption of the nuclear architecture affects the gene expression profile of differentiating neutrophils, and c) to identify the underlying mechanisms that cause disrupted growth and functional responses of neutrophils lacking LBR expression. To achieve these specific aims, experiments will involve the use of immunostaining and fluorescence microscopy to localize expression of nuclear proteins in LBR-deficient cells. Microarrays will be screened for expression of genes in LBR-deficient neutrophils, and possible roles for LBR in mediating cholesterol metabolism in mature neutrophils will be tested. Both in vivo and in vitro transendothelial migration assays will also be used to test for LBR functions in chemotaxis. The results generated by this project will provide the foundation for future studies aimed at further understanding the close relationship between neutrophil nuclear structure and neutrophil function. The results may also offer important insight into the pathogenesis of potentially fatal hematologic disorders. [unreadable] [unreadable] [unreadable]